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Novel Insights Into the Multifaceted Roles of BLM in the Maintenance of Genome Stability

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Novel Insights Into the Multifaceted Roles of BLM in the Maintenance of Genome Stability University of South Florida Scholar Commons Graduate Theses and Dissertations Graduate School April 2019 Novel Insights into the Multifaceted Roles of BLM in the Maintenance of Genome Stability Vivek M. Shastri University of South Florida, [email protected] Follow this and additional works at: https://scholarcommons.usf.edu/etd Part of the Cell Biology Commons, and the Molecular Biology Commons Scholar Commons Citation Shastri, Vivek M., "Novel Insights into the Multifaceted Roles of BLM in the Maintenance of Genome Stability" (2019). Graduate Theses and Dissertations. https://scholarcommons.usf.edu/etd/8413 This Dissertation is brought to you for free and open access by the Graduate School at Scholar Commons. It has been accepted for inclusion in Graduate Theses and Dissertations by an authorized administrator of Scholar Commons. For more information, please contact [email protected]. Novel Insights into the Multifaceted Roles of BLM in the Maintenance of Genome Stability by Vivek M. Shastri A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy with a concentration in Cell and Molecular Biology Department of Cell Biology, Microbiology and Molecular Biology College of Arts and Sciences University of South Florida Major Professor: Kristina H. Schmidt, Ph.D. Alvaro Monteiro, Ph.D. Meera Nanjundan, Ph.D. Sandy Westerheide, Ph.D. Date of Approval: April 5, 2019 Keywords: Bloom syndrome, DNA repair, DNA replication, genome instability Copyright © 2019, Vivek M. Shastri DEDICATION I offer this work to my Guru, Bhagawan Sri Sathya Sai Baba. I dedicate this dissertation to my Mother, Father, and Brother who made it possible for me to pursue my dream. Their hard work and patience have been a constant source of inspiration to me. I especially thank Jennifer, who has always been by my side and encouraged me to better myself. ACKNOWLEDGEMENT First and foremost, I would like to acknowledge Dr. Kristina Schmidt for graciously granting me the opportunity to work in her lab and for being an extremely supportive advisor. I would like to express my gratitude to my committee members for their inputs and patience over the years. Special thanks to past and present members of the lab. I am also thankful for all the help and support from the Department of Cell Biology, Molecular Biology, and Microbiology during my time here. TABLE OF CONTENTS List of Tables ...................................................................................................................................v List of Figures ................................................................................................................................ vi List of Acronyms ......................................................................................................................... viii Abstract .......................................................................................................................................... xi Chapter One: Introduction ..............................................................................................................1 The RecQ Helicase Family .................................................................................................2 The core helicase domain ........................................................................................2 The RecQ C-terminal domain .................................................................................3 The helicase and RNase D–like C-terminal domain ...............................................4 Non-conserved and unique domains .......................................................................5 Human diseases resulting from RecQ helicase deficiencies ...................................8 Rothmund-Thomson syndrome is caused by mutations in RECQL4 ...............................................................................................8 Werner syndrome is caused by defects in WRN ........................................8 BLM deficiency causes Bloom syndrome ..................................................9 Bloom Syndrome .................................................................................................................9 Clinical features of Bloom syndrome .....................................................................9 Cellular defects of Bloom syndrome ....................................................................11 Mutations and Polymorphism in the BLM Gene ...............................................................12 Bloom syndrome causing mutations .....................................................................13 Heterozygosity in BLM and cancer risk ................................................................13 Polymorphism in BLM and disease risk ................................................................15 Functional relevance of coding SNPs in BLM ......................................................15 Structure of the BLM Helicase .........................................................................................17 The BLM C-terminal region contains the helicase core .......................................17 The BLM N-terminal region is predicted to be intrinsically disordered i and forms a major site for protein interactions ...............................................19 Interactions and Functions of BLM ..................................................................................21 Canonical role of BLM in recombinational DNA repair ......................................21 BLM functions in response to DNA replication stress .........................................24 BLM unwinds stable DNA structures that impede DNA replication ...................26 BLM functions in telomere replication and maintenance .....................................27 BLM and ultrafine anaphase bridges ....................................................................28 Regulation of BLM Function ............................................................................................30 Phosphorylation of BLM during the replication stress response ..........................30 Regulation of sub-cellular localization of BLM ...................................................31 PTMs regulate the pro- and anti-recombinogenic activities of BLM ...................32 Regulation of BLM by TopBP1 and MIB1 ..........................................................33 Hypotheses and Aims .......................................................................................................33 References ..........................................................................................................................35 Chapter Two: Cellular Defects Caused by Hypomorphic Variants of the Bloom syndrome Helicase Gene BLM ................................................................................................................55 Abstract .............................................................................................................................55 Background ...........................................................................................................55 Methods and results ..............................................................................................56 Conclusion ............................................................................................................56 Introduction .......................................................................................................................56 Materials and Methods .......................................................................................................60 Cell lines, plasmids, and transfection ...................................................................60 Subcellular fractionation and western blotting .....................................................60 Differential sister‐chromatid staining ...................................................................61 Clonogenic survival assay .....................................................................................61 γH2AX accumulation and elimination ..................................................................62 Comet assay ..........................................................................................................62 Results ...............................................................................................................................63 Discussion .........................................................................................................................68 Future Directions ..............................................................................................................73 Figures and Tables ............................................................................................................76 References .........................................................................................................................89 ii Chapter Three: Generation and Characterization of a Diploid BLM Knockout Cell Line ...........95 Introduction .......................................................................................................................95 Materials and Methods ......................................................................................................97 Cell lines, plasmid constructs and transfections ...................................................97 CRISPR/Cas9-mediated BLM knockout ..............................................................97
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